Two enzymes, a cyclic-AMP-dependent protein kinase and a phosphoprotein phosphatase, have been purified to near homogeneity from bovine heart. A suitable substrate has been sought so that the kinetics of a monocyclic cascade system can be defined and understood. Three potential substrates have been purified for this purpose. Histone H1 was purified to homogeneity, but was found to activate the protein kinase. Attempts to modify the H1 and eliminate this activation effect were only partially successful. Consequently, the use of H1 had to be abandoned. A different protein with one phosphorylatable site is liver pyruvate kinase. This enzyme was purified from beef liver, but could not be phosphorylated; the reason for this is not yet clear. Human erythrocyte pyruvate kinase was also partially purified as a potential substrate for the phosphorylation-dephosphorylation cascade. While this work was in progress, a novel substrate was discovered; the 20,000 dalton light chain of chicken gizzard myosin. In order to assess the applicability of this to a model cascade system, and because it may be important in the regulation of smooth muscle contraction, the protein chemistry and kinetics of myosin light chain phosphorylation by protein kinase were characterized.